THE 2.46-ANGSTROM RESOLUTION STRUCTURE OF THE PANCREATIC LIPASE-COLIPASE COMPLEX INHIBITED BY A C-11 ALKYL PHOSPHONATE

Pancreatic lipase belongs to the serine esterase family and can theref ore be inhibited by classical serine reagents such as diisopropyl fluo ride or E600. In an attempt to further characterize the active site an d catalytic mechanism, we synthesized a C11 alkyl phosphonate compound . This compound is an effective inhibitor of pancreatic lipase. The cr ystal structure of the pancreatic lipase-colipase complex inhibited by this compound was determined at a resolution of 2.46 Angstrom and ref ined to a final R-factor of 18.3%. As was observed in the case of the structure of the ternary pancreatic lipase-colipase-phospholipid compl ex, the binding of the ligand induces rearrangements of two surface lo ops in comparison with the closed structure of the enzyme (van Tilbeur gh et al., 1993b). The inhibitor, which could be clearly observed in t he active site, was covalently bound to the active site serine Ser152. A racemic mixture of the inhibitor was used in the crystallization, a nd there exists evidence that both enantiomers are bound at the active site. The C11 alkyl chain of the first enantiomer fits into a hydroph obic groove and is thought to thus mimic the interaction between the l eaving fatty acid of a triglyceride substrate and the protein, The alk yl chain of the second enantiomer also has an elongated conformation a nd interacts with hydrophobic patches on the surface of the open amphi pathic lid. This may indicate the location of a second alkyl chain of a triglyceride substrate, Some of the detergent molecules, needed for the crystallization, were also observed in the crystal. Some of them w ere located at the entrance of the active site, bound to the hydrophob ic part of the lid. On the basis of this crystallographic study, a hyp othesis about the binding mode of real substrates and the organization of the active site is proposed.